The present invention relates to a novel bacterial species. The novel species is most similar to the genus Paracoccus, as was determined by 16S ribosomal RNA analysis. However, the novel bacterial species produces and actively secretes carotenoid containing vesicles, during at least some stages of its life cycle, a phenomenon yet unseen, and therefore, the novel bacterial species, most likely, represents the first isolate of a new genus. The present invention further relates to a process for production of carotenoids, such as, but not limited to, .beta.-carotene, echinenone, .beta.-cryptoxanthin, canthaxanthin, adonirubin, cis-adonixanthin, adonixanthin, astaxanthin and zeaxanthin, using a species producing and secreting same.
The carotenoids of the present invention are natural pigments useful as feed additives, food additives, cosmetics, etc. As further detailed below, especially, astaxanthin is valuable from an industrial point of view as a feed additive, such as a color improver, for bred fishes such as salmon, trout, red sea bream etc., and as a safe natural food additive. In addition, adonixanthin is, if its industrial production process is established, promising as both food and feed additive as the astaxanthin is.
Further, .beta.-carotene has been used as a food additive, feed additive, pharmaceutical substance, etc. Echinenone is promised as a food additive, feed additive, etc. Canthaxanthin has been used as a food additive, feed additive, in cosmetics etc. And zeaxanthin has been used as a food additive, feed additive, etc.
Carotenoids are natural pigments that are responsible for many of the yellow, orange and red colors seen in living organisms. Carotenoids are widely distributed in nature and have, in various living systems, two main biological functions. They serve as light-harvesting pigments in photosynthesis, and they protect against photooxidative damage. These and additional biological functions of carotenoids, their important industrial role, their biosynthesis and organisms producing them are discussed hereinbelow.
As part of the light-harvesting antenna, carotenoids can absorb photons and transfer the energy to chlorophyll, thus assisting in the harvesting of light in the range of 450-570 nm see, Cogdell R. J. and Frank H. A. (1987) How carotenoids function in photosynthetic bacteria. Biochim Biophys Acta 895: 63-79; Cogdell R. (1988) The function of pigments in chloroplasts. In: Goodwin T. W. (ed) Plant Pigments, pp 183-255. Academic Press, London; Frank H. A., Violette C. A., Trautman J. K., Shreve A. P., Owens T. G. and Albrecht A. C. (1991) Carotenoids in photosynthesis: structure and photochemistry. Pure Appl Chem 63: 109-114; Frank H. A., Farhoosh R., Decoster B. and Christensen R. L. (1992) Molecular features that control the efficiency of carotenoid-to-chlorophyll energy transfer in photosynthesis. In: Murata N. (ed) Research in Photosynthesis, Vol I, pp 125-128. Kluwer, Dordrecht; and, Cogdell R. J. and Gardiner A. T. (1993) Functions of carotenoids in photosynthesis. Meth Enzymol 214: 185-193!. Although carotenoids are integral constituents of the protein-pigment complexes of the light-harvesting antennae in photosynthetic organisms, they are also important components of the photosynthetic reaction centers.
Most of the total carotenoids is located in the light harvesting complex II Bassi R., Pineaw B., Dainese P. and Marquartt J. (1993) Carotenoid binding proteins of photosystem II. Eur J Biochem 212: 297-302!. The identities of the photosynthetically active carotenoproteins and their precise location in light-harvesting systems are not known. Carotenoids in photochemically active chlorophyll-protein complexes of the thermophilic cyanobacterium Synechococcus sp. were investigated by linear dichroism spectroscopy of oriented samples see, Breton J. and Kato S. (1987) Orientation of the pigments in photosystem II: low-temperature linear-dichroism study of a core particle and of its chlorophyll-protein subunits isolated from Synechococcus sp. Biochim Biophys Acta 892: 99-107!. Those complexes contained mainly a .beta.-carotene pool absorbing around 505 and 470 nm, which is oriented close to the membrane plane. In photochemically inactive chlorophyll-protein complexes, the .beta.-carotene absorbs around 495 and 465 nm, and the molecules are oriented perpendicular to the membrane plane.
Evidence that carotenoids are associated with cyanobacterial photosystem (PS) II has been described see, Suzuki R. and Fujita Y. (1977) Carotenoid photobleaching induced by the action of photosynthetic reaction center II: DCMU sensitivity. Plant Cell Physiol 18: 625-631; and, Newman P. J. and Sherman L. A. (1978) Isolation and characterization of photosystem I and II membrane particles from the blue-green alga Synechococcus cedrorum. Biochim Biophys Acta 503: 343-361!. There are two .beta.-carotene molecules in the reaction center core of PS II see, Ohno T., Satoh K. and Katoh S. (1986) Chemical composition of purified oxygen-evolving complexes from the thermophilic cyanobacterium Synechococcus sp. Biochim Biophys Acta 852: 1-8; Gounaris K., Chapman D. J. and Barber J. (1989) Isolation and characterization of a D1/D2/cytochrome b-559 complex from Synechocystis PCC6803. Biochim Biophys Acta 973: 296-301; and, Newell R. W., van Amerongen H, Barber J. and van Grondelle R. (1993) Spectroscopic characterization of the reaction center of photosystem II using polarized light: Evidence for .beta.-carotene excitors in PS II reaction centers. Biochim Biophys Acta 1057: 232-238! whose exact function(s) is still obscure reviewed by Satoh K. (1992) Structure and function of PS II reaction center. In: Murata N. (ed) Research in Photosynthesis, Vol. II, pp. 3-12. Kluwer, Dordrecht!. It was demonstrated that these two coupled .beta.-carotene molecules protect chlorophyll P680 from photodamage in isolated PS II reaction centers see, De Las Rivas J., Telfer A. and Barber J. (1993) 2-coupled .beta.-carotene molecules protect P680 from photodamage in isolated PS II reaction centers. Biochim. Biophys. Acta 1142: 155-164!, and this may be related to the protection against degradation of the D1 subunit of PS II see, Sandmann G. (1993) Genes and enzymes involved in the desaturation reactions from phytoene to lycopene. (abstract), 10th International Symposium on Carotenoids, Trondheim CL1-2!. The light-harvesting pigments of a highly purified, oxygen-evolving PS II complex of the thermophilic cyanobacterium Synechococcus sp. consists of 50 chlorophyll a and 7 .beta.-carotene, but no xanthophyll, molecules see, Ohno T., Satoh K. and Katoh S. (1986) Chemical composition of purified oxygen-evolving complexes from the thermophilic cyanobacterium Synechococcus sp. Biochim Biophys Acta 852: 1-8!. .beta.-carotene was shown to play a role in the assembly of an active PS II in green algae see, Humbeck K., Romer S. and Senger H. (1989) Evidence for the essential role of carotenoids in the assembly of an active PS II. Planta 179: 242-250!.
Isolated complexes of PS I from Phormidium luridum, which contained 40chlorophylls per P700, contained an average of 1.3 molecules of .beta.-carotene see, Thornber J. P., Alberte R. S., Hunter F. A., Shiozawa J. A. and Kan K. S. (1976) The organization of chlorophyll in the plant photosynthetic unit. Brookhaven Symp Biology 28: 132-148!. In a preparation of PS I particles from Synechococcus sp. strain PCC 6301, which contained 130.+-.5 molecules of antenna chlorophylls per P700, 16 molecules of carotenoids were detected see, Lundell D. J., Glazer A. N., Melis A. and Malkin R. (1985) Characterization of a cyanobacterial photosystem I complex. J Biol Chem 260: 646-654!. A substantial content of .beta.-carotene and the xanthophylls cryptoxanthin and isocryptoxanthin were detected in PS I pigment-protein complexes of the thermophilic cyanobacterium Synechococcus elongatus see, Coufal J., Hladik J. and Sofrova D. (1989) The carotenoid content of photosystem 1 pigment-protein complexes of the cyanobacterium Synechococcus elongatus. Photosynthetica 23: 603-616!. A subunit protein-complex structure of PS I from the thermophilic cyanobacterium Synechococcus sp., which consisted of four polypeptides (of 62, 60, 14 and 10 kDa), contained approximately 10 .beta.-carotene molecules per P700 see, Takahashi Y., Hirota K. and Katoh S. (1985) Multiple forms of P700-chlorophyll a-protein complexes from Synechococcus sp.: the iron, quinone and carotenoid contents. Photosynth Res 6: 183-192!. This carotenoid is exclusively bound to the large polypeptides which carry the functional and antenna chlorophyll a. The fluorescence excitation spectrum of these complexes suggested that .beta.-carotene serves as an efficient antenna for PS I.
As mentioned, an additional essential function of carotenoids is to protect against photooxidation processes in the photosynthetic apparatus that are caused by the excited triplet state of chlorophyll. Carotenoid molecules with .pi.-electron conjugation of nine or more carbon-carbon double bonds can absorb triplet-state energy from chlorophyll and thus prevent the formation of harmful singlet-state oxygen radicals. In Synechococcus sp. the triplet state of carotenoids was monitored in closed PS II centers and its rise kinetics of approximately 25 nanoseconds is attributed to energy transfer from chlorophyll triplets in the antenna see, Schlodder E. and Brettel K. (1988) Primary charge separation in closed photosystem II with a lifetime of 11 nanoseconds. Flash-absorption spectroscopy with oxygen-evolving photosystem II complexes from Synechococcus. Biochem Biophys Acta 933: 22-34!. It is conceivable that this process, that has a lower yield compared to the yield of radical-pair formation, plays a role in protecting chlorophyll from damage due to over-excitation.
The protective role of carotenoids in vivo has been elucidated through the use of bleaching herbicides such as norflurazon that inhibit carotenoid biosynthesis in all organisms performing oxygenic photosynthesis reviewed by Sandmann G. and Boger P. (1989) Inhibition of carotenoid biosynthesis by herbicides. In: Boger P. and Sandmann G. (Eds.) Target Sites of Herbicide Action, pp 25-44. CRC Press, Boca Raton, Fla.!. Treatment with norfilurazon in the light results in a decrease of both carotenoid and chlorophyll levels, while in the dark, chlorophyll levels are unaffected. Inhibition of photosynthetic efficiency in cells of Oscillatoria agardhii that were treated with the pyridinone herbicide, fluridone, was attributed to a decrease in the relative abundance of myxoxanthophyll, zeaxanthin and .beta.carotene, which in turn caused photooxidation of chlorophyll molecules see, Canto de Loura I., Dubacq J. P. and Thomas J. C. (1987) The effects of nitrogen deficiency on pigments and lipids of cianobacteria. Plant Physiol 83: 838-843!.
It has been demonstrated in plants that zeaxanthin is required to dissipate, in a nonradiative manner, the excess excitation energy of the antenna chlorophyll see, Demmig-Adams B. (1990) Carotenoids and photoprotection in plants: a role for the xanthophyll zeaxanthin. Biochim Biophys Acta 1020: 1-24; and, Demmig-Adams B. and Adams W. W. III (1990) The carotenoid zeaxanthin and high-energy-state quenching of chlorophyll fluorescence. Photosynth Res 25: 187-197!. In algae and plants a light-induced deepoxidation of violaxanthin to yield zeaxanthin, is related to photoprotection processes reviewed by Demmig-Adams B. and Adams W. W. III (1992) Photoprotection and other responses of plants to high light stress. Ann Rev Plant Physiol Plant Mol Biol 43: 599-626!. The light-induced deepoxidation of violaxanthin and the reverse reaction that takes place in the dark, are known as the "xanthophyll cycle" see, Demmig-Adams B. and Adams W. W. III (1992) Photoprotection and other responses of plants to high light stress. Ann Rev Plant Physiol Plant Mol Biol 43: 599-626!. Cyanobacterial lichens, that do not contain any zeaxanthin and that probably are incapable of radiation energy dissipation, are sensitive to high light intensity; algal lichens that contain zeaxanthin are more resistant to high-light stress see, Demmig-Adams B., Adams W. W. III, Green T. G. A., Czygan F. C. and Lange O. L. (1990) Differences in the susceptibility to light stress in two lichens forming a phycosymbiodeme, one partner possessing and one lacking the xanthophyll cycle. Oecologia 84: 451-456; Demmig-Adams B. and Adams W. W. III (1993) The xanthophyll cycle, protein turnover, and the high light tolerance of sun-acclimated leaves. Plant Physiol 103: 1413-1420; and, Demmig-Adams B. (1990) Carotenoids and photoprotection in plants: a role for the xanthophyll zeaxanthin. Biochim Biophys Acta 1020: 1-24!. In contrast to algae and plants, cyanobacteria do not have a xanthophyll cycle. However, they do contain ample quantities of zeaxanthin and other xanthophylls that can support photoprotection of chlorophyll.
Several other functions have been ascribed to carotenoids. The possibility that carotenoids protect against damaging species generated by near ultra-violet (UV) irradiation is suggested by results describing the accumulation of .beta.-carotene in a UV-resistant mutant of the cyanobacterium Gloeocapsa alpicola see, Buckley C. E. and Houghton J. A. (1976) A study of the effects of near UV radiation on the pigmentation of the blue-green alga Gloeocapsa alpicola. Arch Microbiol 107: 93-97!. This has been demonstrated more elegantly in Escherichia coli against phototoxic produce carotenoids see, Tuveson R. W. and Sandmann G. (1993) Protection by cloned carotenoid genes expressed in Escherichia coli against phototoxic molecules activated by near-ultraviolet light. Meth Enzymol 214: 323-330!. Due to their ability to quench oxygen radical species, carotenoids are efficient antioxidants and thereby protect cells from oxidative damage. This function of carotenoids is important in virtually all organisms see, Krinsky N. I. (1989) Antioxidant functions of carotenoids. Free Radical Biol Med 7: 617-635; and, Palozza P. and Krinsky N. I. (1992) Antioxidant effects of carotenoids in vivo and in vitro--an overview. Meth Enzymol 213: 403-420!. Other cellular functions could be affected by carotenoids, even if indirectly.
Although carotenoids in cyanobacteria are not the major photoreceptors for phototaxis, an influence of carotenoids on phototactic reactions, that have been observed in Anabaena variabilis, was attributed to the removal of singlet oxygen radicals that may act as signal intermediates in this system see, Nultsch W. and Schuchart H. (1985) A model of the phototactic reaction chain of cyanobacterium Anabaena variabilis. Arch Microbiol 142: 180-184!.
In flowers and fruits carotenoids facilitate the attraction of pollinators and dispersal of seeds. This latter aspect is strongly associated with agriculture. The type and degree of pigmentation in fruits and flowers are among the most important traits of many crops. This is mainly since the colors of these products often determine their appeal to the consumers and thus can increase their market worth.
Carotenoids have important commercial uses as coloring agents in the food industry since they are non-toxic see, Bauernfeind J. C. (1981) Carotenoids as colorants and vitamin A precursors. Academic Press, London!. The red color of the tomato fruit is provided by lycopene which accumulates during fruit ripening in chromoplasts. Tomato extracts, which contain high content (over 80% dry weight) of lycopene, are commercially produced worldwide for industrial use as food colorant. Furthermore, the flesh, feathers or eggs of fish and birds assume the color of the dietary carotenoid provided, and thus carotenoids are frequently used in dietary additives for poultry and in aquaculture. Certain cyanobacterial species for example Spirulina sp. see, Sommer T. R., Potts W. T. and Morrissy N. M. (1990) Recent progress in processed microalgae in aquaculture. Hydrobiologia 204/205: 435-443!, are cultivated in aquaculture for the production of animal and human food supplements. Consequently, the content of carotenoids, primarily of .beta.-carotene, in these cyanobacteria has a major commercial implication in biotechnology.
Most carotenoids are composed of a C.sub.40 hydrocarbon backbone, constructed from eight C.sub.5 isoprenoid units and contain a series of conjugated double bonds. Carotenes do not contain oxygen atoms and are either linear or cyclized molecules containing one or two end rings. Xanthophylls are oxygenated derivatives of carotenes. Various glycosilated carotenoids and carotenoid esters have been identified. The C.sub.40 backbone can be further extended to give C.sub.45 or C.sub.50 carotenoids, or shortened yielding apocarotenoids. Some nonphotosynthetic bacteria also synthesize C.sub.30 carotenoids. General background on carotenoids can be found in Goodwin T. W. (1980) The Biochemistry of the Carotenoids, Vol. 1, 2nd Ed. Chapman and Hall, New York; and in Goodwin T. W. and Britton G. (1988) Distribution and analysis of carotenoids. In: Goodwin T. W. (ed) Plant Pigments, pp 62-132. Academic Press, New York.
More than 640 different naturally-occurring carotenoids have been so far characterized, hence, carotenoids are responsible for most of the various shades of yellow, orange and red found in microorganisms, fungi, algae, plants and animals. Carotenoids are synthesized by all photosynthetic organisms as well as several nonphotosynthetic bacteria and fungi, however they are also widely distributed through feeding throughout the animal kingdom.
Carotenoids are synthesized de novo from isoprenoid precursors only in photosynthetic organisms and some microorganisms, they typically accumulate in protein complexes in the photosynthetic membrane, in the cell membrane and in the cell wall.
As detailed in FIG. 1, in the biosynthesis pathway of .beta.-carotene, four enzymes convert geranylgeranyl pyrophosphate of the central isoprenoid pathway to .beta.-carotene. Carotenoids are produced from the general isoprenoid biosynthetic pathway. While this pathway has been known for several decades, only recently, and mainly through the use of genetics and molecular biology, have some of the molecular mechanisms involved in carotenoids biogenesis, been elucidated. This is due to the fact that most of the enzymes which take part in the conversion of phytoene to carotenes and xanthophylls are labile, membrane-associated proteins that lose activity upon solubilization see, Beyer P., Weiss G. and Kleinig H. (1985) Solubilization and reconstitution of the membrane-bound carotenogenic enzymes from daffodile chromoplasts. Eur J Biochem 153: 341-346; and, Bramley P. M. (1985) The in vitro biosynthesis of carotenoids. Adv Lipid Res 21: 243-279!.
However, solubilization of carotenogenic enzymes from Synechocystis sp. strain PCC 6714 that retain partial activity has been reported see, Bramley P. M. and Sandmann G. (1987) Solubilization of carotenogenic enzyme of Aphanocapsa. Phytochem 26: 1935-1939!.
There is no genuine in vitro system for carotenoid biosynthesis which enables a direct essay of enzymatic activities. A cell-free carotenogenic system has been developed see, Clarke I. E., Sandmann G., Bramley P. M. and Boger P. (1982) Carotene biosynthesis with isolated photosynthetic membranes. FEBS Lett 140: 203-206! and adapted for cyanobacteria see, Sandmann G. and Bramley P. M. (1985) Carotenoid biosynthesis by Aphanocapsa homogenates coupled to a phytoene-generating system from Phycomyces blakesleeanus. Planta 164: 259-263; and, Bramley P. M. and Sandmann G. (1985) In vitro and in vivo biosynthesis of xanthophylls by the cyanobacterium Aphanocapsa. Phytochem 24: 2919-2922!.
Reconstitution of phytoene desaturase from Synechococcus sp. strain PCC 7942 in liposomes was achieved following purification of the polypeptide, that had been expressed in Escherichia coli see, Fraser P. D., Linden H. and Sandmann G. (1993) Purification and reactivation of recombinant Synechococcus phytoene desaturase from an overexpressing strain of Escherichia coli. Biochem J 291: 687-692!.
Referring again to FIG. 1, carotenoids are synthesized from isoprenoid precursors. The central pathway of isoprenoid biosynthesis may be viewed as beginning with the conversion of acetyl-CoA to mevalonic acid. D.sup.3 -isopentenyl pyrophosphate (IPP), a C.sub.5 molecule, is formed from mevalonate and is the building block for all long-chain isoprenoids. Following isomerization of IPP to dimethylallyl pyrophosphate (DMAPP), three additional molecules of IPP are combined to yield the C.sub.20 molecule, geranylgeranyl pyrophosphate (GGPP). These 1'-4 condensation reactions are catalyzed by prenyl transferases see, Kleinig H. (1989) The role of plastids in isoprenoid biosynthesis. Ann Rev Plant Physiol Plant Mol Biol 40: 39-59!. There is evidence in plants that the same enzyme, GGPP synthase, carries out all the reactions from DMAPP to GGPP see, Dogbo O. and Camara B. (1987) Purification of isopentenyl pyrophosphate isomerase and geranylgeranyl pyrophosphate synthase from Capsicum chromoplasts by affinity chromatography. Biochim Biophys Acta 920: 140-148; and, Laferriere A. and Beyer P. (1991) Purification of geranylgeranyl diphosphate synthase from Sinapis alba etioplasts. Biochim Biophys Acta 216: 156-163!.
The first step that is specific for carotenoid biosynthesis is the head-to-head condensation of two molecules of GGPP to produce prephytoene pyrophosphate (PPPP). Following removal of the pyrophosphate, GGPP is converted to 15-cis-phytoene, a colorless C.sub.40 hydrocarbon molecule. This two-step reaction is catalyzed by the soluble enzyme, phytoene synthase, an enzyme encoded by a single gene (crtB), in both cyanobacteria and plants see, Chamovitz D., Misawa N., Sandmann G. and Hirschberg J. (1992) Molecular cloning and expression in Escherichia coli of a cyanobacterial gene coding for phytoene synthase, a carotenoid biosynthesis enzyme. FEBS Lett 296: 305-310; Ray J. A., Bird C. R., Maunders M., Grierson D. and Schuch W. (1987) Sequence of pTOM5, a ripening related cDNA from tomato. Nucl Acids Res 15: 10587-10588; Camara B. (1993) Plant phytoene synthase complex--component 3 enzymes, immunology, and biogenesis. Meth Enzymol 214: 352-365!. All the subsequent steps in the pathway occur in membranes. Four desaturation (dehydrogenation) reactions convert phytoene to lycopene via phytofluene, .zeta.-carotene, and neurosporene. Each desaturation increases the number of conjugated double bonds by two such that the number of conjugated double bonds increases from three in phytoene to eleven in lycopene.
Relatively little is known about the molecular mechanism of the enzymatic dehydrogenation of phytoene see, Jones B. L. and Porter J. W. (1986) Biosynthesis of carotenes in higher plants. CRC Crit Rev Plant Sci 3: 295-324; and, Beyer P., Mayer M. and Kleinig H. (1989) Molecular oxygen and the state of geometric iosomerism of intermediates are essential in the carotene desaturation and cyclization reactions in daffodil chromoplasts. Eur J Biochem 184: 141-150!. It has been established that in cyanobacteria, algae and plants the first two desaturations, from 15-cis-phytoene to .zeta.-carotene, are catalyzed by a single membrane-bound enzyme, phytoene desaturase see, Jones B. L. and Porter J. W. (1986) Biosynthesis of carotenes in higher plants. CRC Crit Rev Plant Sci 3: 295-324; and, Beyer P., Mayer M. and Kleinig H. (1989) Molecular oxygen and the state of geometric iosomerism of intermediates are essential in the carotene desaturation and cyclization reactions in daffodil chromoplasts. Eur J Biochem 184: 141-150!. Since the .zeta.-carotene product is mostly in the all-trans configuration, a cis-trans isomerization is presumed at this desaturation step. The primary structure of the phytoene desaturase polypeptide in cyanobacteria is conserved (over 65% identical residues) with that of algae and plants see, Pecker I., Chamovitz D., Linden H., Sandmann G. and Hirschberg J. (1992) A single polypeptide catalyzing the conversion of phytoene to .zeta.-carotene is transcriptionally regulated during tomato fruit ripening. Proc Natl Acad Sci USA 89: 4962-4966; Pecker I. Chamovitz D., Mann V., Sandmann G., Boger P. and Hirschberg J. (1993) Molecular characterization of carotenoid biosynthesis in plants: the phytoene desaturase gene in tomato. In: Murata N. (ed) Research in Photosynthesis, Vol III, pp 11-18. Kluwer, Dordrectht!. Moreover, the same inhibitors block phytoene desaturase in the two systems see, Sandmann G. and Boger P. (1989) Inhibition of carotenoid biosynthesis by herbicides. In: Boger P. and Sandmann G. (eds) Target Sites of Herbicide Action, pp 25-44. CRC Press, Boca Raton, Fla.!. Consequently, it is very likely that the enzymes catalyzing the desaturation of phytoene and phytofluene in cyanobacteria and plants have similar biochemical and molecular properties, that are distinct from those of phytoene desaturases in other microorganisms. One such a difference is that phytoene desaturases from Rhodobacter capsulatus, Erwinia sp. or fungi convert phytoene to neurosporene, lycopene, or 3,4-dehydrolycopene, respectively.
Desaturation of phytoene in daffodil chromoplasts see, Beyer P., Mayer M. and Kleinig H. (1989) Molecular oxygen and the state of geometric iosomerism of intermediates are essential in the carotene desaturation and cyclization reactions in daffodil chromoplasts. Eur J Biochem 184: 141-150!, as well as in a cell free system of Synechococcus sp. strain PCC 7942 see, Sandmann G. and Kowalczyk S. (1989) In vitro carotenogenesis and characterization of the phytoene desaturase reaction in Anacystis. Biochem Biophys Res Com 163: 916-921!, is dependent on molecular oxygen as a possible final electron acceptor, although oxygen is not directly involved in this reaction. A mechanism of dehydrogenase-electron transferase was supported in cyanobacteria over dehydrogenation mechanism of dehydrogenase-monooxygenase see, Sandmann G. and Kowalczyk S. (1989) In vitro carotenogenesis and characterization of the phytoene desaturase reaction in Anacystis. Biochem Biophys Res Com 163: 916-921!. A conserved FAD-binding motif exists in all phytoene desaturases whose primary structures have been analyzed see, Pecker I., Chamovitz D., Linden H., Sandmann G. and Hirschberg J. (1992) A single polypeptide catalyzing the conversion of phytoene to .zeta.-carotene is transcriptionally regulated during tomato fruit ripening. Proc Natl Acad Sci USA 89: 4962-4966; Pecker I., Chamovitz D., Mann V., Sandmann G., Boger P. and Hirschberg J. (1993) Molecular characterization of carotenoid biosynthesis in plants: the phytoene desaturase gene in tomato. In: Murata N. (ed) Research in Photosynthesis, Vol III, pp 11-18. Kluwer, Dordrectht!. The phytoene desaturase enzyme in pepper was shown to contain a protein-bound FAD see, Hugueney P., Romer S., Kuntz M. and Camara B. (1992) Characterization and molecular cloning of a flavoprotein catalyzing the synthesis of phytofluene and .zeta.-carotene in Capsicum chromoplasts. Eur J Biochem 209: 399-407!. Since phytoene desaturase is located in the membrane, an additional, soluble redox component is predicted. This hypothetical component could employ NAD(P).sup.+, as suggested see, Mayer M. P., Nievelstein V. and Beyer P. (1992) Purification and characterization of a NADPH dependent oxidoreductase from chromoplasts of Narcissus pseudonarcissus--a redox-mediator possibly involved in carotene desaturation. Plant Physiol Biochem 30: 389-398! or another electron and hydrogen carrier, such as a quinone. The cellular location of phytoene desaturase in Synechocystis sp. strain PCC 6714 and Anabaena variabilis strain ATCC 29413 was determined with specific antibodies to be mainly (85%) in the photosynthetic thylakoid membranes see, Serrano A., Gimenez P., Schmidt A. and Sandmann G. (1990) Immunocytochemical localization and functional determination of phytoene desaturase in photoautotrophic prokaryotes. J Gen Microbiol 136: 2465-2469!.
In cyanobacteria algae and plants .zeta.-carotene is converted to lycopene via neurosporene. Very little is known about the enzymatic mechanism, which is predicted to be carried out by a single enzyme see, Linden H., Vioque A. and Sandmann G. (1993) Isolation of a carotenoid biosynthesis gene coding for .zeta.carotene desaturase from Anabaena PCC 7120 by heterologous complementation. FEMS Microbiol Lett 106: 99-104!. The deduced amino acid sequence of .zeta.-carotene desaturase in Anabaena sp. strain PCC 7120 contains a dinucleotide-binding motif that is similar to the one found in phytoene desaturase.
Two cyclization reactions convert lycopene to .beta.-carotene. Evidence has been obtained that in Synechococcus sp. strain PCC 7942 see, Cunningham F. X. Jr, Chamovitz D., Misawa N., Gantt E. and Hirschberg J. (1993) Cloning and functional expression in Escherichia coli of a cyanobacterial gene for lycopene cyclase, the enzyme that catalyzes the biosynthesis of .beta.-carotene. FEBS Lett 328: 130-138!, as well as in plants see, Camara B. and Dogbo O. (1986) Demonstration and solubilization of lycopene cyclase from Capsicum chromoplast membranes. Plant Physiol 80: 172-184!, these two cyclizations are catalyzed by a single enzyme, lycopene cyclase. This membrane-bound enzyme is inhibited by the triethylamine compounds, CPTA and MPTA see, Sandmann G. and Boger P. (1989) Inhibition of carotenoid biosynthesis by herbicides. In: Boger P. and Sandmann G. (eds) Target Sites of Herbicide Action, pp 25-44. CRC Press, Boca Raton, Fla.!. Cyanobacteria carry out only the .beta.-cyclization and therefore do not contain .epsilon.-carotene, .delta.-carotene and .alpha.-carotene and their oxygenated derivatives. The .beta.-ring is formed through the formation of a "carbonium ion" intermediate when the C-1,2 double bond at the end of the linear lycopene molecule is folded into the position of the C-5,6 double bond, followed by a loss of a proton from C-6. No cyclic carotene has been reported in which the 7,8 bond is not a double bond. Therefore, full desaturation as in lycopene, or desaturation of at least half-molecule as in neurosporene, is essential for the reaction. Cyclization of lycopene involves a dehydrogenation reaction that does not require oxygen. The cofactor for this reaction is unknown. A dinucleotide-binding domain was found in the lycopene cyclase polypeptide of Synechococcus sp. strain PCC 7942, implicating NAD(P) or FAD as coenzymes with lycopene cyclase.
The addition of various oxygen-containing side groups, such as hydroxy-, methoxy-, oxo-, epoxy-, aldehyde or carboxylic acid moieties, form the various xanthophyll species. Little is known about the formation of xanthophylls. Hydroxylation of .beta.-carotene requires molecular oxygen in a mixed-function oxidase reaction.
Clusters of genes encoding the enzymes for the entire pathway have been cloned from the purple photosynthetic bacterium Rhodobacter capsulatus see, Armstrong G. A., Alberti M., Leach F. and Hearst J. E. (1989) Nucleotide sequence, organization, and nature of the protein products of the carotenoid biosynthesis gene cluster of Rhodobacter capsulatus. Mol Gen Genet 216: 254-2681 and from the nonphotosynthetic bacteria Erwinia herbicola see, Sandmann G., Woods W. S. and Tuveson R. W. (1990) Identification of carotenoids in Erwinia herbicola and in transformed Escherichia coli strain. FEMS Microbiol Lett 71: 77-82; Hundle B. S., Beyer P., Kleinig H., Englert H. and Hearst J. E. (1991) Carotenoids of Erwinia herbicola and an Escherichia coli HB101 strain carrying the Erwinia herbicola carotenoid gene cluster. Photochem Photobiol 54: 89-93; and, Schnurr G., Schmidt A. and Sandmann G. (1991) Mapping of a carotenogenic gene cluster from Erwinia herbicola and functional identification of six genes. FEMS Microbiol Lett 78: 157-162! and Erwinia uredovora see, Misawa N., Nakagawa M., Kobayashi K., Yamano S., Izawa I., Nakamura K. and Harashima K. (1990) Elucidation of the Erwinia uredovora carotenoid biosynthetic pathway by functional analysis of gene products in Escherichia coli. J Bacteriol 172: 6704-6712!. Two genes, al-3 for GGPP synthase see, Nelson M. A., Morelli G., Carattoli A., Romano N. and Macino G. (1989) Molecular cloning of a Neurospora crassa carotenoid biosynthetic gene (albino-3) regulated by blue light and the products of the white collar genes. Mol Cell Biol 9: 1271-1276; and, Carattoli A., Romano N., Ballario P, Morelli G. and Macino G. (1991) The Neurospora crassa carotenoid biosynthetic gene (albino 3). J Biol Chem 266: 5854-5859! and al-1 for phytoene desaturase see, Schmidhauser T. J., Lauter F. R., Russo V. E. A. and Yanofsky C. (1990) Cloning sequencing and photoregulation of al-1, a carotenoid biosynthetic gene of Neurospora crassa. Mol Cell Biol 10: 5064-5070! have been cloned from the fungus Neurospora crassa. However, attempts at using these genes as heterologous molecular probes to clone the corresponding genes from cyanobacteria or plants were unsuccessful due to lack of sufficient sequence similarity.
The first "plant-type" genes for carotenoid synthesis enzyme were cloned from cyanobacteria using a molecular-genetics approach. In the first step towards cloning the gene for phytoene desaturase, a number of mutants that are resistant to the phytoene-desaturase-specific inhibitor, norflurazon, were isolated in Synechococcus sp. strain PCC 7942 see, Linden H., Sandmann G., Chamovitz D., Hirschberg J. and Boger P. (1990) Biochemical characterization of Synechococcus mutants selected against the bleaching herbicide norflurazon. Pestic Biochem Physiol 36: 46-51!. The gene conferring norflurazon-resistance was then cloned by transforming the wild-type strain to herbicide resistance see, Chamovitz D., Pecker I. and Hirschberg J. (1991) The molecular basis of resistance to the herbicide norflurazon. Plant Mol Biol 16: 967-974; Chamovitz D., Pecker I., Sandmann G., Boger P. and Hirschberg J. (1990) Cloning a gene for norflurazon resistance in cyanobacteria. Z Naturforsch 45c: 482-486!. Several lines of evidence indicated that the cloned gene, formerly called pds and now named crtP, codes for phytoene desaturase. The most definitive one was the functional expression of phytoene desaturase activity in transformed Escherichia coli cells see, Linden H., Misawa N., Chamovitz D., Pecker I., Hirschberg J. and Sandmann G. (1991) Functional complementation in Escherichia coli of different phytoene desaturase genes and analysis of accumulated carotenes. Z Naturforsch 46c: 1045-1051; and, Pecker I. Chamovitz D., Linden H., Sandmann G. and Hirschberg J. (1992) A single polypeptide catalyzing the conversion of phytoene to .zeta.-carotene is transcriptionally regulated during tomato fruit ripening. Proc Natl Acad Sci USA 89: 4962-4966!. The crtP gene was also cloned from Synechocystis sp. strain PCC 6803 by similar methods see, Martinez-Ferez I. M. and Vioque A. (1992) Nucleotide sequence of the phytoene desaturase gene from Synechocystis sp. PCC 6803 and characterization of a new mutation which confers resistance to the herbicide norflurazon. Plant Mol Biol 18: 981-983!.
The cyanobacterial crtP gene was subsequently used as a molecular probe for cloning the homologous gene from an alga see, Pecker I., Chamovitz D., Mann V., Sandmann G., Boger P. and Hirschberg J. (1993) Molecular characterization of carotenoid biosynthesis in plants: the phytoene desaturase gene in tomato. In: Murata N. (ed) Research in Photosynthesis, Vol III, pp 11-18. Kluwer, Dordrectht! and higher plants see, Bartley G. E., Viitanen P. V., Pecker I., Chamovitz D., Hirschberg J. and Scolnik P. A. (1991) Molecular cloning and expression in photosynthetic bacteria of a soybean cDNA coding for phytoene desaturase, an enzyme of the carotenoid biosynthesis pathway. Proc Natl Acad Sci USA 88: 6532-6536; and, Pecker I., Chamovitz D., Linden H., Sandmann G. and Hirschberg J. (1992) A single polypeptide catalyzing the conversion of phytoene to .zeta.-carotene is transcriptionally regulated during tomato fruit ripening. Proc Natl Acad Sci USA 89: 4962-4966!. The phytoene desaturases in Synechococcus sp. strain PCC 7942 and Synechocystis sp. strain PCC 6803 consist of 474 and 467 amino acid residues, respectively, whose sequences are highly conserved (74% identities and 86% similarities). The calculated molecular mass is 51 kDa and, although it is slightly hydrophobic (hydropathy index -0.2), it does not include a hydrophobic region which is long enough to span a lipid bilayer membrane. The primary structure of the cyanobacterial phytoene desaturase is highly conserved with the enzyme from the green alga Dunalliela bardawil (61% identical and 81% similar; see, Pecker I., Chamovitz D., Mann V., Sandmann G., Boger P. and Hirschberg J. (1993) Molecular characterization of carotenoid biosynthesis in plants: the phytoene desaturase gene in tomato. In: Murata N. (ed) Research in Photosynthesis, Vol III, pp 11-18. Kluwer, Dordrectht!) and from tomato see, Pecker I., Chamovitz D., Linden H., Sandmann G. and Hirschberg J. (1992) A single polypeptide catalyzing the conversion of phytoene to .zeta.-carotene is transcriptionally regulated during tomato fruit ripening. Proc Natl Acad Sci USA 89: 4962-4966!, pepper see, Hugueney P., Romer S., Kuntz M. and Camara B. (1992) Characterization and molecular cloning of a flavoprotein catalyzing the synthesis of phytofluene and .zeta.-carotene in Capsicum chromoplasts. Eur J Biochem 209: 399-407! and soybean see, Bartley G. E., Viitanen P. V., Pecker I., Chamovitz D., Hirschberg J. and Scolnik P. A. (1991) Molecular cloning and expression in photosynthetic bacteria of a soybean cDNA coding for phytoene desaturase, an enzyme of the carotenoid biosynthesis pathway. Proc Natl Acad Sci USA 88: 6532-6536! (62-65% identical and .about.79% similar; see, Chamovitz D. (1993) Molecular analysis of the early steps of carotenoid biosynthesis in cyanobacteria: Phytoene synthase and phytoene desaturase. Ph.D. Thesis, The Hebrew University of Jerusalem!). The eukaryotic phytoene desaturase polypeptides are larger (64 kDa); however, they are processed during import into the plastids to mature forms whose sizes are comparable to those of the cyanobacterial enzymes.
There is a high degree of structural similarity in carotenoid enzymes of Rhodobacter capsulatus, Erwinia sp. and Neurospora crassa reviewed in Armstrong G. A., Hundle B. S. and Hearst J. E. (1993) Evolutionary conservation and structural similarities of carotenoid biosynthesis gene products from photosynthetic and nonphotosynthetic organisms. Meth Enzymol 214: 297-311!, including in the crtI gene-product, phytoene desaturase. As indicated above, a high degree of conservation of the primary structure of phytoene desaturases also exists among oxygenic photosynthetic organisms. However, there is little sequence similarity, except for the FAD binding sequences at the amino termini, between the "plant-type" crtP gene products and the "bacterial-type" phytoene desaturases (crtI gene products; 19-23% identities and 42-47% similarities). It has been hypothesized that crtP and crtI are not derived from the same ancestral gene and that they originated independently through convergent evolution see, Pecker I, Chamovitz D., Linden H., Sandmann G. and Hirschberg J. (1992) A single polypeptide catalyzing the conversion of phytoene to .zeta.-carotene is transcriptionally regulated during tomato fruit ripening. Proc Natl Acad Sci USA 89: 4962-4966!. This hypothesis is supported by the different dehydrogenation sequences that are catalyzed by the two types of enzymes and by their different sensitivities to inhibitors.
Although not as definite as in the case of phytoene desaturase, a similar distinction between cyanobacteria and plants on the one hand and other microorganisms is also seen in the structure of phytoene synthase. The crtB gene (formerly psy) encoding phytoene synthase was identified in the genome of Synechococcus sp. strain PCC 7942 adjacent to crtP and within the same operon see, Bartley G. E., Viitanen P. V., Pecker I., Chamovitz D., Hirschberg J. and Scolnik P. A. (1991) Molecular cloning and expression in photosynthetic bacteria of a soybean cDNA coding for phytoene desaturase, an enzyme of the carotenoid biosynthesis pathway. Proc Natl Acad Sci USA 88: 6532-6536!. This gene encodes a 36-kDa polypeptide of 307 amino acids with a hydrophobic index of -0.4. The deduced amino acid sequence of the cyanobacterial phytoene synthase is highly conserved with the tomato phytoene synthase (57% identical and 70% similar; Ray J. A., Bird C. R., Maunders M., Grierson D. and Schuch W. (1987) Sequence of pTOM5, a ripening related cDNA from tomato. Nucl Acids Res 15: 10587-10588!) but is less highly conserved with the crtB sequences from other bacteria (29-32% identical and 48-50% similar with ten gaps in the alignment). Both types of enzymes contain two conserved sequence motifs also found in prenyl transferases from diverse organisms see, Bartley G. E., Viitanen P. V., Pecker I., Chamovitz D., Hirschberg J. and Scolnik P. A. (1991) Molecular cloning and expression in photosynthetic bacteria of a soybean cDNA coding for phytoene desaturase, an enzyme of the carotenoid biosynthesis pathway. Proc Natl Acad Sci USA 88: 6532-6536; Carattoli A., Romano N., Ballario P, Morelli G. and Macino G. (1991) The Neurospora crassa carotenoid biosynthetic gene (albino 3). J Biol Chem 266: 5854-5859; Armstrong G. A., Hundle B. S. and Hearst J. E. (1993) Evolutionary conservation and structural similarities of carotenoid biosynthesis gene products from photosynthetic and nonphotosynthetic organisms. Meth Enzymol 214: 297-311; Math S. K., Hearst J. E. and Poulter C. D. (1992) The crtE gene in Erwinia herbicola encodes geranylgeranyl diphosphate synthase. Proc Natl Acad Sci USA 89: 6761-6764; and, Chamovitz D. (1993) Molecular analysis of the early steps of carotenoid biosynthesis in cyanobacteria: Phytoene synthase and phytoene desaturase. Ph.D. Thesis, The Hebrew University of Jerusalem!. It is conceivable that these regions in the polypeptide are involved in the binding and/or removal of the pyrophosphate during the condensation of two GGPP molecules.
The crtQ gene encoding .zeta.-carotene desaturase (formerly zds) was cloned from Anabaena sp. strain PCC 7120 by screening an expression library of cyanobacterial genomic DNA in cells of Escherichia coli carrying the Erwinia sp. crtB and crtE genes and the cyanobacterial crtp gene see, Linden H., Vioque A. and Sandmann G. (1993) Isolation of a carotenoid biosynthesis gene coding for .zeta.-carotene desaturase from Anabaena PCC 7120 by heterologous complementation. FEMS Microbiol Lett 106: 99-104!. Since these Escherichia coli cells produce .zeta.carotene, brownish-red pigmented colonies that produced lycopene could be identified on the yellowish background of cells producing .zeta.-carotene. The predicted .zeta.-carotene desaturase from Anabaena sp. strain PCC 7120 is a 56-kDa polypeptide which consists of 499 amino acid residues. Surprisingly, its primary structure is not conserved with the "plant-type" (crP gene product) phytoene desaturases, but it has considerable sequence similarity to the bacterial-type enzyme (crtI gene product) see, Sandmann G. (1993) Genes and enzymes involved in the desaturation reactions from phytoene to lycopene. (abstract), 10th International Symposium on Carotenoids, Trondheim CL1-2!. It is possible that the cyanobacterial crtQ gene and crtI gene of other microorganisms originated in evolution from a common ancestor.
The crtL gene for lycopene cyclase (formerly Lcy) was cloned from Synechococcus sp. strain PCC 7942 utilizing essentially the same cloning strategy as for crtP. By using an inhibitor of lycopene cyclase, 2-(4-methylphenoxy)triethylamine hydrochloride (MPTA), the gene was isolated by transformation of the wild-type to herbicide-resistance see, Cunningham F. X. Jr, Chamovitz D., Misawa N., Gantt E. and Hirschberg J. (1993) Cloning and functional expression in Escherichia coli of a cyanobacterial gene for lycopene cyclase, the enzyme that catalyzes the biosynthesis of .beta.-carotene. FEBS Lett 328: 130-138!. Lycopene cyclase is the product of a single gene product and catalyzes the double cyclization reaction of lycopene to .beta.-carotene. The crtL gene product in Synechococcus sp. strain PCC 7942 is a 46-kDa polypeptide of 411 amino acid residues. It has no sequence similarity to the crtY gene product (lycopene cyclase) from Erwinia uredovora or Erwinia herbicola.
The gene for .beta.-carotene hydroxylase (crtZ) and zeaxanthin glycosilase (crtX) have been cloned from Erwinia herbicola see, Hundle B., Alberti M., Nievelstein V., Beyer P., Kleinig H., Armstrong G. A., Burke D. H. and Hearst J. E. (1994) Functional assignment of Erwinia herbicola Eho10 carotenoid genes expressed in Escherichia coli. Mol Gen Genet 254: 406-416; Hundle B. S., Obrien D. A., Alberti M., Beyer P. and Hearst J. E. (1992) Functional expression of zeaxanthin glucosyltransferase from Erwinia herbicola and a proposed diphosphate binding site. Proc Natl Acad Sci USA 89: 9321-9325! and from Erwinia uredovora see, Misawa N., Nakagawa M., Kobayashi K., Yamano S., Izawa I., Nakamura K. and Harashima K. (1990) Elucidation of the Erwinia uredovora carotenoid biosynthetic pathway by functional analysis of gene products in Escherichia coli. J Bacteriol 172: 6704-6712!.
The unicellular fresh-water green alga Haematococcus pluvialis accumulates large amounts of (3S,3'S) astaxanthin when exposed to unfavorable growth conditions, or following different environmental stresses such as phosphate or nitrogen starvation, high concentration of salt in the growth medium or high light intensity see, Yong Y. Y. R. and Lee Y. K. (1991) Phycologia 30 257-261; Droop M. R. (1954) Arch Microbiol 20: 391-397; and, Andrewes A. G., Borch G., Liaaen-Jensen S. and Snatzke G. (1974) Acta Chem Scand B28: 730-736!. During this process, the vegetative cells of the alga form cysts and change their color from green to red. A cDNA from Haematococcus pluvialis, designated crtO, which encodes a .beta.-C-4-oxygenase, the enzyme that converts .beta.-carotene to canthaxanthin, and its expression in a heterologous systems expressing .beta.-carotene hydroxylase (e.g., Erwinia herbicola crtZ gene product), leading to the production of (3S,3'S) astaxanthin are described in Harker M., Hirschberg J. (1997) Biosynthesis of ketocarotenoids in transgenic cyanobacteria expressing the algal gene for beta-C-4-oxygenase, crtO. FEBS Lett. 404:129-134.
The ketocarotenoid astaxanthin (3,3'-dihydroxy-.beta.,.beta.-carotene-4,4'-dione) was first described in aquatic crustaceans as an oxidized form of .beta.-carotene. Astaxanthin was later found to be very common in many marine animals and algae. However, only few animals can synthesize astaxanthin de novo from other carotenoids and most of them obtain it in their food. In the plant kingdom, astaxanthin occurs mainly in some species of cyanobacteria, algae and lichens. However, it is found rarely also in petals of higher plant species see, Goodwin T. W. (1980) The Biochemistry of the carotenoids, Vol. 1. 2nd Ed, Chapman and Hall, London and New York!. FIG. 2 presents the biosynthesis pathway of astaxanthin.
The function of astaxanthin as a powerful antioxidant in animals has been demonstrated see, Miki W. (1991) Biological functions and activities of animal carotenoids. Pure Appl Chem 63: 141!. Astaxanthin is a strong inhibitor of lipid peroxidation and has been shown to play an active role in the protection of biological membranes from oxidative injury see, Palozza P. and Krinsky N. I. (1992) Antioxidant effects of carotenoids in vivo and in vitro--an overview. Methods Enzymol 213: 403-420; and, Kurashige M., Okimasu E., Inove M. and Utsumi K. (1990) Inhibition of oxidative injury of biological membranes by astaxanthin. Physiol Chem Phys Med NMR 22: 27!. The chemopreventive effects of astaxanthin have also been investigated in which astaxanthin was shown to significantly reduce the incidence of induced urinary bladder cancer in mice see, Tanaka T., Morishita Y., Suzui M., Kojima T., Okumura A. and Mori H. (1994). Chemoprevention of mouse urinary bladder carcinogenesis by the naturally occurring carotenoid astaxanthin. Carcinogenesis 15: 15!. It has also been demonstrated that astaxanthin exerts immunomodulating effects by enhancing antibody production see, Jyonouchi H., Zhang L. and Tomita Y. (1993) Studies of immunomodulating actions of carotenoids. II. Astaxanthin enhances in vitro antibody production to T-dependent antigens without facilitating polyclonal B-cell activation. Nutr Cancer 19: 269; and, Jyonouchi H., Hill J. R., Yoshifumi T. and Good R. A. (1991) Studies of immunomodulating actions of carotenoids. I. Effects of .beta.-carotene and astaxanthin on murine lymphocyte functions and cell surface marker expression in-vitro culture system. Nutr Cancer 16: 93!. The complete biomedical properties of astaxanthin remain to be elucidated, but initial results suggest that it could play an important role in cancer and tumor prevention, as well as eliciting a positive response from the immune system.
Astaxanthin is the principal carotenoid pigment of salmonids and shrimps and imparts attractive pigmentation in the eggs, flesh and skin see, Torrisen O. J., Hardy R. W., Shearer K. D. (1989) Pigmentation of salmonid-carotenoid deposition and metabolism in salmonids. Crit Rev Aquatic Sci 1: 209!. The world-wide harvest of salmon in 1991 was approximately 720,000 MT., of which 25-30% were produced in a variety of aquaculture facilities see, Meyers S. P. (1994) Developments in world aquaculture, feed formulations, and role of carotenoids. Pure Appl Chem 66: 1069!. This is set to increase up to 460,000 MT. by the year 2000 see, Bjorndahl T. (1990) The Economics of Salmon Aquaculture. Blackwell Scientific, Oxford. pp. 1!. The red coloration of the salmonid flesh contributes to consumer appeal and therefore affects the price of the final product. Animals cannot synthesize carotenoids and they acquire the pigments through the food chain from the primary producers--marine algae and phytoplankton. Those grown in intensive culture usually suffer from suboptimal color. Consequently, carotenoid-containing nourishment is artificially added in aquaculture, at considerable cost to the producer.
Astaxanthin is the most expensive commercially used carotenoid compound (todays-1995 market value is of 2,500-3,500 $/kg). It is utilized mainly as nutritional supplement which provides pigmentation in a wide variety of aquatic animals. In the Far-East it is used also for feeding poultry to yield a typical pigmentation of chickens. It is also a desirable and effective nontoxic coloring for the food industry and is valuable in cosmetics. Recently it was reported that astaxanthin is a potent antioxidant in humans and thus is a desirable food additive.
Natural (3S,3'S) astaxanthin is limited in availability. It is commercially extracted from some crustacea species see, Torrisen O. J., Hardy R. W., Shearer K. D. (1989) Pigmentation of salmonid-carotenoid deposition and metabolism in salmonids. Crit Rev Aquatic Sci 1: 209!. The (3R,3'R) stereoisomer of astaxanthin is produced from Phaffia a yeast specie, see, Andrewes A. G., Phaff H. J. and Starr M. P. (1976) Carotenoids of Phaffia rhodozyma, a red-pigmented fermenting yeast. Phytochemistry Vol. 15, pp. 1003-1007!. Synthetic astaxanthin, comprising a 1:2:1 mixture of the (3S,3'S)-, (3S,3'R)- and (3R,3'R)-isomers is now manufactured by Hoffman-La Roche and sold at a high price (ca. $2,500/Kg) under the name "CAROPHYLL PINK" see, Mayer H. (1994) Reflections on carotenoid synthesis. Pure & Appl Chem, Vol. 66, pp. 931-938!. Recently a novel gene involved in ketocompound biosynthesis, designated crtW was isolated from the marine bacteria Agrobacterium auranticacum and Alcaligenes PC-1 that produce ketocarotenoids such as astaxanthin. When the crtW gene was introduced into engineered Eschrichia coli that accumulated .beta.-carotene due to Erwinia carotenogenic genes, the Escherichia coli transformants synthesized canthaxanthin a precursor in the synthetic pathway of astaxanthin see, Misawa N., Kajiwara S., Kondo K., Yokoyama A., Satomi Y., Saito T., Miki W. and Ohtani T. (1995) Canthaxanthin biosynthesis by the conversion of methylene to keto groups in a hydrocarbon .beta.-carotene by a single gene. Biochemical and biophysical research communications Vol. 209, pp. 867-876!. It is therefore desirable to find a relatively inexpensive source of (3S,3'S) astaxanthin to be used as a feed supplement in aquaculture and as a valuable chemical for various other industrial uses.
It is known that astaxanthin is contained in fishes such as red sea bream, salmon, trout, etc., and Crustacea such as shrimps, crabs, crawfishes and krills Carotenoids of Marine Organisms; edt. Nippon Suisan Gakukai, 1978!. As microorganisms which produce astaxanthin, red yeast, Phaffla rhodozyma Phytochemistry, 15, 1009, 1976!, Brevibacterium Journal of General and Applied Microbiology, 15, 127, 1969!, and green algea Haematococcus pluvialis Phytochemistry, 20, 2561, 1981! are known. As chemical Synthetic processes, conversion of .beta.-carotene Pure Appl. Chem. 57, 741, 1985! and synthesis from phosphonium salt Helv. Chim. Acta. 64, 2436, 1981! are known.
However, the known process for production of astaxanthin are not advantageous due to high cost, because the content of astaxanthin in natural products such as krills, crawfishes, etc. is very low, and extraction thereof is difficult. In addition, stable availability of the resources is problematic. Moreover, slow growth rate and low astaxanthin productivity of the red yeast, Phaffla rhodozyma, makes this source of ketocarotenoids impractical from the industrial point of view.
The growth rate of the green algea, Haematococcus pluvialis, is also low, the culture is easily contaminated, and extraction of astaxanthin is extremely difficult because of the thick cell wall of this alga. Therefore, industrial production of astaxanthin from algea is difficult.
It is known that adonixanthin is contained in goldfishes and carps Carotenoids of Marine Organisms, Nippon Suisan Gakukai, 1978!, but it is believed that chemical synthesis of adonixanthin is difficult. No industrial process for production of adonixanthin is known.
As production processes for .beta.-carotene, although synthesis from .beta.-Ionone Pure & Appl. Chem. 63(1), 45, 1991!, and extraction from green or yellow vegetables such as carrot, sweet potato, pumpkin, etc., are known Natural Coloring Agent Handbook, Korin, 1979, edt. by editorial committee of Natural Coloring Agent Handbook!, production cost of these processes is high.
As processes for production of .beta.-carotene by microorganisms, the production by an algea Dunaliella J. Appl. Bacteriol., 70, 181, 1991!, and the production by a fungus Blakeslea J. Appl. Bacteriol., 70, 181, 1991! are known. Production of .beta.-carotene by bacteria is also known for a certain bacterial species described in U.S. Pat. No. 5,607,839.
Echinenone is extracted from natural products, for example, starfishes such as crown-of-thorns starfish, the internal organs of fishes such as red sea bream, sea urchin, the internal organs of Crustacea such as lobster, etc. Carotenoids of Marine Organisms, edt. Nippon Suisan Gakukai, 1987!. However, the production of echinenone by microorganisms is known only for a species of an unknown genus, described in U.S. Pat. No. 5,607,839.
Canthaxanthin is known to be contained in several species of mushrooms Botanical Gazette, 112, 228-232, 1950!, fishes, Crustacea etc. Carotenoids of Marine Organisms, edt. Nippon Suisan Gakukai, 1978!.
The production of echinenone by microorganisms are exemplified by the production by microorganisms belonging to the genus Brevibacterium Applied and Environmental Microbiology, 55(10), 2505, 1989!, and by microorganisms belonging to the genus Rhodococcus Japanese Patent Publication No. 2-138996!. In addition, as chemical synthetic processes, oxidation of .beta.-carotene J. Amer. Chem. Soc., 78, 1427, 1956! and synthesis from novel compound 3-oxo-C.sub.15 phosphonium salt Pure & Appl. Chem. 51, 875, 1979! are known.
As processes for production of zeaxanthin, a chemical synthesis starting from a hydroxy ketone obtained by asymmetric reduction of oxoisophorone Pure & Appl. Chem., 63(1), 45, 1991!, extraction from corn seeds Scitai Shikiso, 1974, Asakura Shoten!, and a process using Flavobacterium Carotenoids, In Microbial Technology, 2nd edn. Vol. 1, 529-544, New York: Academic Press! are known.
There is thus a widely recognized need for, and it would be highly advantageous to have, carotenoids, such as astaxanthin, producing and secreting bacteria, since such bacteria offer a readily available source of carotenoids.
Other features and advantages of the invention will become apparent from the following description and from the claims.